(1) Field of the Invention
The present invention relates to methods for reducing detectable mannosylation of N-linked and O-linked oligosaccharides in yeast. In particular, the present invention provides recombinant yeast host cells in which expression of the GDP-mannose transporter encoded by the Vanadate Resistant Glycosylation 4 (VRG4) gene has been disrupted. In general, the VRG4 gene is essential for cell viability; however, the present invention provides host cells that are viable when expression of the VRG4 gene therein has been disrupted.
(2) Description of Related Art
The ability to produce recombinant human proteins has led to major advances in human health care and remains an active area of drug discovery. Many therapeutic proteins require the posttranslational addition of glycans to specific asparagine residues (N-glycosylation) of the protein to ensure proper structure-function activity and subsequent stability in human serum. For therapeutic use in humans, glycoproteins require human-like N-glycosylation. Mammalian cell lines (e.g., CHO cells, human retinal cells) that can mimic human-like glycoprotein processing have several drawbacks including low protein titers, long fermentation times, heterogeneous products, and continued viral containment. It is therefore desirable to use an expression system that not only produces high protein titers with short fermentation times, but can also produce human-like glycoproteins.
Fungal hosts such as the methylotrophic yeast Pichia pastoris have distinct advantages for therapeutic protein expression, for example, they do not secrete high amounts of endogenous proteins, strong inducible promoters for producing heterologous proteins are available, they can be grown in defined chemical media and without the use of animal sera, and they can produce high titers of recombinant proteins (Cregg et al., FEMS Microbiol. Rev. 24: 45-66 (2000)). However, glycosylated proteins expressed in P. pastoris generally contain additional mannose sugars resulting in “high mannose” glycans, as well as mannosylphosphate groups which impart a negative charge onto glycoproteins. Glycoproteins with either high mannose glycans or charged mannans present the risk of eliciting an unwanted immune response in humans (Takeuchi, Trends in Glycosci. Glycotechnol. 9:S29-S35 (1997); Rosenfeld and Ballou, J. Biol. Chem. 249: 2319-2321 (1974)). Accordingly, it is desirable to produce therapeutic glycoproteins in fungal host cells wherein the pattern of glycosylation on the glycoprotein is identical to or similar to that which occurs on glycoproteins produced in humans and which do not have detectable yeast glycosylation.